Bioluminescence in beetles is characterized by a widerange of colors. Fireflies (Lampyridae) emit in the green-yellow region of the spectrum (1, 2), click-beetles (Elateridae) emit in the green-orange (2, 3), but railroad-worms (Phengodidae) span the widest range of the spectrum, that is, from the green to the red region (4, 5). The emission of green-red light was suggested to be an adaptation to optimize the detection of bioluminescence in distinct photic environments and for different biological functions (6). In all cases, such distinct colors arise from structurally homologous luciferases, which catalyze the same ATP-dependent oxidation of D-luciferin (7). Most studies about the structure and function of beetle luciferases have focused on a set of luciferases arising mainly from fireflies (8-14), two click-beetle species (15, 16), and recently a North American species of Phengodes (17). Three main factors at the level of the luciferase active site are believed to govern bioluminescence colors (7): (I) the presence of basic residues catalyzing tautomerization between a ketonic (red light emitter) and enolic (yellow-green light emitter) species of excited oxy-luciferin (18-20); (II) the hydrophobicity of the active site (21, 22); and (III) the active site conformation which affects rotation of excited oxyluciferin along the C2-C2′ bond (23). These factors may act together or independently to determine distinct bioluminescence colors in luciferases of different species. The construction of chimeric proteins using click-beetle luciferases (24) and firefly luciferases (25), along with mutagenesis studies (26-28), have revealed important regions and key residues for the bioluminescence color determination. The crystallographic structure of firefly luciferase has been recently resolved in the absence of the substrates (29), which shows a main N-terminal domain and a smaller C-terminal cleft which supposedly come closer to sandwich the substrates during catalysis. Despite all these studies, no structural investigations had been conducted on naturally occurring red light-emitting luciferases.
The beautiful and rare Phrixothrix railroad-worms are probably the most spectacular luminescent beetles, because in addition to their yellow-green bioluminescence (λmax=542-574 nm), displayed by two sets of 11 dorsal-lateral lanterns along the body, they emit red bioluminescence (λmax=609-638 nm) through cephalic and postcephalic organs (4, 5), a unique property among terrestrial creatures. The function of the lateral lantern bioluminescence is probably associated with defensive and sexual attraction purposes, whereas in the case of the red lantern bioluminescence was associated with self-illumination (5); however experimental evidence is still lacking. Due to their scarcity, only preliminary biochemical studies have been conducted about these creature luciferases (4, 20). Railroad-worm and click-beetle luciferases share a common feature: they do not suffer batchromic shift upon decreasing pH as lampyrid luciferases do (20). Due to their peculiar spectral properties, Phrixothrix luciferases constitute very important models for understanding the mechanism of color modulation in beetle bioluminescence.